Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

Targeting AMPK-ULK1-mediated autophagy for combating BET inhibitor resistance in acute myeloid leukemia stem cells

Bromodomain and extraterminal domain (BET) inhibitors are promising epigenetic agents for the treatment of various subsets of acute myeloid leukemia (AML). However, the resistance of leukemia stem cells (LSCs) to BET inhibitors remains a major challenge. In this study, we evaluated the mechanisms underlying LSC resistance to the BET inhibitor JQ1. We evaluated the levels of apoptosis and macroautophagy/autophagy induced by JQ1 in LSC-like leukemia cell lines and primary CD34⁺ CD38^? leukemic blasts obtained from AML cases with normal karyotype without recurrent mutations. JQ1 effectively induced apoptosis in a concentration-dependent manner in JQ1-sensitive AML cells. However, in JQ1-resistant AML LSCs, JQ1 induced little apoptosis and led to upregulation of BECN1/Beclin 1, increased LC3 lipidation, formation of autophagosomes, and downregulation of SQSTM1/p62. Inhibition of autophagy by pharmacological inhibitors or knockdown of BECN1 using specific siRNA enhanced JQ1-induced apoptosis in resistant cells, indicating that prosurvival autophagy occurred in these cells. Independent of MTOR signaling, activation of the AMPK (p-Thr172)-ULK1 (p-Ser555) pathway was found to be associated with JQ1-induced autophagy in resistant cells. AMPK inhibition using the pharmacological inhibitor compound C or by knockdown of PRKAA/AMPKα suppressed autophagy and promoted JQ1-induced apoptosis in AML LSCs. These findings revealed that prosurvival autophagy was one of the mechanisms involved in the resistance of AML LSCs to JQ1. Targeting the AMPK-ULK1 pathway or inhibition of autophagy could be an effective therapeutic strategy for combating resistance to BET inhibitors in AML and other types of cancer.     

Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^?/? mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells

We have previously shown that elevated expression of mitotic kinase aurora kinase A (AURKA) in cancer cells promotes the development of metastatic phenotypes and is associated clinically with adverse prognosis. Here, we first revealed a clinically positive correlation between AURKA and autophagy-associated protein SQSTM1 in breast cancer and further demonstrated that AURKA regulated SQSTM1 through autophagy. Indeed, depletion by siRNA or chemical inhibition of AURKA by the small molecule VX-680 increased both the level of microtubule-associated protein 1 light chain 3-II (LC3-II) and the number of autophagosomes, along with decreased SQSTM1. Conversely, overexpression of AURKA inhibited autophagy, as assessed by decreased LC3-II and increased SQSTM1 either upon nutrient deprivation or normal conditions. In addition, phosphorylated forms of both RPS6KB1 and mechanistic target of rapamycin (MTOR) were elevated by overexpression of AURKA whereas they were suppressed by depletion or inhibition of AURKA. Moreover, inhibition of MTOR by PP242, an inhibitor of MTOR complex1/2, abrogated the changes in both LC3-II and SQSTM1 in AURKA-overexpressing BT-549 cells, suggesting that AURKA-suppressed autophagy might be associated with MTOR activation. Lastly, repression of autophagy by depletion of either LC3 or ATG5, sensitized breast cancer cells to VX-680-induced apoptosis. Similar findings were observed in cells treated with the autophagy inhibitors chloroquine (CQ) and bafilomycin A₁ (BAF). Our data thus revealed a novel role of AURKA as a negative regulator of autophagy, showing that AURKA inhibition induced autophagy, which may represent a novel mechanism of drug resistance in apoptosis-aimed therapy for breast cancer.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA⁺ but not VacA^- strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

ADIPOQ/adiponectin induces cytotoxic autophagy in breast cancer cells through STK11/LKB1-mediated activation of the AMPK-ULK1 axis

ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.     

A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.     

Research Article: Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^−/− mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.     

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint

Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47^PHOX (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA (+) but not VacA (-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.